Methods for reducing the complexity of DNA sequence

ABSTRACT

Genomic or cDNA, or fragments and mixtures thereof, can be screened by generation of subsets and then subjecting the subsets to mismatch scanning procedures. Alternatively, DNA fragments can be generated by cutting with a restriction endonuclease that generates variable overhangs. For either of the above methods, Y-shaped adapters having a region of non-complementary single-stranded DNA at the end can be used. Heterohybrid DNA, containing one DNA strand derived from each of two different samples, or homohybrids, containing DNA strands from the same sample, can be selected. Adapters attached to the ends of the fragments are designed to allow the selective isolation of homohybrid or heterohybrid DNA.

This application is a divisional of U.S. Ser. No. 09/398,217, filed Sep.17, 1999, still pending.

THIS APPLICATION CLAIMS THE BENEFIT OF APPLICATION SER. NO. 60/100,999FILED SEP. 18, 1998 Technical Field of the Invention

This invention relates to methods for reducing the complexity of DNAmixtures, subsequent analysis of genetic variations, and isolation ofprobes or clones of regions of interest.

Background of the Invention

In 1993 Nelson and associates described a “genomic mismatch scanning”(GMS) method to directly identify identical-by-descent (IBD) sequencesin yeast (Nelson, S. F., et al, Nature Genetics, 1993, 4:11-18; this andother papers, books and patents cited herein are expressly incorporatedin their entireties by reference). The method allows DNA fragments fromIBD regions between two relatives to be isolated based on their abilityto form mismatch-free hybrid molecules. The method consists of digestingDNA fragments from two sources with a restriction endonuclease thatproduces protruding 3′-ends. The protruding 3′-ends provide someprotection from exonuclease III (Exo III), which is used in later steps.The two sources are distinguished by methylating the DNA from only onesource. Molecules from both sources are denatured and reannealed,resulting in the formation of four types of duplex molecules:homohybrids formed from strands derived from the same source andheterohybrids consisting of DNA strands from different sources.Heterohybrids can either be mismatch-free or contain base-pairmismatches, depending on the extent of identity of homologous regions.

Homohybrids are distinguished from heterohybrids by use of restrictionendonucleases that cleave fully methylated or unmethylated GATC sites.Homohybrids are cleaved into smaller duplex molecules. Heterohybridscontaining a mismatch are distinguished from mismatch-free molecules byuse of the E. coli methyl-directed mismatch repair system. Thecombination of three proteins of the methyl-directed mismatch repairsystem MutS, MutL, and MutH (herein collectively called MutSLH) alongwith ATP introduce a single-strand nick on the unmethylated strand atGATC sites in duplexes that contain a mismatch (Welsh, et al., J Biol.Chem., 1987, 262:15624). Heterohybrids that do not contain a mismatchare not nicked. All molecules are then subjected to digestion by ExoIII, which can initiate digestion at a nick, a blunt end, or a recessed3′-end, to produce single-stranded gaps. Only mismatch-freeheterohybrids are not subject to attack by Exo III; all other moleculeshave single-stranded gaps introduced by the enzyme. Molecules withsingle-stranded regions are removed by absorption to benzoylatednapthoylated DEAE cellulose. The remaining molecules consist ofmismatch-free heterohybrids which may represent regions of IBD.

Nelson, et al., used S. cerevisiae hybrids as a model system and showedthat sequences shared by two independently generated hybrids from thesame parent strains could be identified in many instances. Experimentsof this kind are much easier to do in yeast than in humans. The yeastgenome is 250 times simpler than the human genome, it contains far fewerrepetitive sequences, and genomic sequences of two yeast strains differmore than genomes of unrelated humans. It has thus far not been possibleto do comparable experiments with human genomic DNA. In order to do soone needs to use methods to reproducibly generate simplified but highlypolymorphic representations of the human genome. Pooling techniquesbased on mathematical principles are also essential to identify IBDsequences as well as other sequences showing allele frequencydifferences (AFD) (Shaw, S. H., et al., Genome Research, Cold SpringHarbor Laboratory Press, 1998, 8:111-123).

The human genome is enormously long, at 3×10⁹ base pairs, and it is fartoo complex for efficient reannealing of homologous DNA strands afterdenaturation. The rate of annealing of a mixture of nucleic acidfragments in liquid phase is inversely proportional to the square oftheir complexity. Efforts have therefore been made to generatesimplified representations of the genome for genetic methods based oncross hybridization of homologous sequences from different genomes. Theexact degree of simplification of human genomic DNA needed to achieveefficient annealing depends on the conditions of hybridization includingtotal DNA concentration, hybridization buffer, and temperature. Ingeneral a 10-100 fold simplification is needed for efficient annealingto occur at high DNA concentrations in high salt aqueous solutions(Lisitsyn, N. A., et al., Science, 1993, 259:946-951).

In some embodiments of the invention, DNA sequences of interest arereplicated in rolling circle amplification reactions (RCA). RCA is anisothermal amplification reaction in which a DNA polymerase extends aprimer on a circular template (Komberg, A. and Baker, T. A., DNAReplication, W. H. Freeman, New York, 1991). The product consists oftandemly linked copies of the complementary sequence of the template.RCA can be used as a DNA amplification method (Fire, A. and Si-Qun Xu,Proc. Natl. Acad. Sci. USA, 1991, 92:4641-4645; Lui, D., et al. J Am.Chem. Soc., 1995, 118:1587-1594; Lizardi, P. M., et al., NatureGenetics, 1998, 19:225-232). RCA can also be used in a detection methodusing a probe called a “padlock probe” (Nilsson, M., et al., NatureGenetics, 1997, 16: 252-255).

It would be useful to have superior ways of analyzing human DNA andother complex DNA samples.

SUMMARY OF THE INVENTION

A general method for screening genomic or cDNA, or fragments andmixtures thereof, involves sample simplification by the generation ofsubsets and then subjecting the subsets to mismatch scanning procedures.Any given DNA sequence will be represented in one and only one subset,minimizing the number of subsets required to detect a sequence ofinterest and guaranteeing that all possible sequences can potentially becovered by analyzing all possible subsets. The complexity of DNAsequences is reduced by attaching adapters to the ends of DNA fragmentsthat allow the specific subsets of DNA to be selected and amplified. Insome procedures, subsets are generated by replicating DNA in apolymerase chain reaction (PCR) or single primer extension reactionsusing primers that are complementary to sequences in the adapter andwhich, at the 3′-end, are complementary to a subset of sequences in thegenomic or cDNA.

In another version of this method, DNA fragments are generated bycutting with a restriction endonuclease, such as Bsl1, that generatesvariable overhangs for which some of the nucleotides can have any of 2to 4 of the bases A, C, G, or T. In this case, subsets are generated byligating adapters to the fragment ends that have a specific sequence inthe overhang and a primer binding site unique for each adapter. Foreither of the above methods, Y-shaped adapters can be used having aregion of non-complementary single-stranded DNA at the end. Therefore,following ligation, the DNA fragment-plus-adapter construct has thenon-complementary region at its ends. Use of Y-shaped adapters make itpossible to generate non-overlapping subsets such that a given DNAfragment will only be represented in one of the possible subsets.

Procedures are given for isolating selected subsets from other,contaminating DNAs by using primers that have attached chemical moietiesthat can be captured on beads, columns, and the like. In some cases, theDNA is then released by cutting specifically designed sequences in theprimers with restriction endonucleases. Fragment DNA is protected fromthese restriction endonucleases by methylation. The DNA subsets obtainedare sufficiently reduced in complexity to allow improved analysis ofsequence polymorphism by mismatch scanning procedures. Procedures aregiven for selecting DNA fragments representing regions of lowpolymorphism or for generating fragments depleted for regions of lowpolymorphism.

In some embodiments, the DNA fragments are replicated in a rollingcircle amplification procedure (RCA; see reviews by Hingorani, M. M.,and O'Donnell, M., Current Biology, 1998, 8:R83-86 and by Kelman, Z., etal., Structure, 1998, 6:121-5). The DNA polymerase III holoenzyme(hereafter sometimes denoted DNA pol III) is used in most of thesemethods to increase the rate and processivity of primer extension. DNApol III also improves the ability to replicate through a DNA region ofhigh GC content or other obstructions that tend to block DNApolymerases.

A method is also given for selecting heterohybrid DNA that contains oneDNA strand derived from each of two different samples or homohybrids inwhich the DNA strands from different samples have not been recombined.Each DNA sample may consist of some concentration of a unique DNAfragment, or a mixture of fragments, and each sample may be derived froma single individual or more than one individual. The different DNAsamples are mixed together, denatured, and then reannealed. Some of theDNA strands will reanneal back together with another strand from thesame DNA sample forming a homohybrid. Other DNA strands will reannealwith a DNA strand from a different sample forming a heterohybrid.Adapters attached to the ends of the fragments are designed to allow theselective isolation of homohybrid or heterohybrid DNA. In one method,restriction endonuclease recognition sites are present in the adapterssuch that homohybrid or heterohybrid DNA can be selectively eliminateddepending on the ability of the restriction endonuclease to cut the DNA.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a diagram illustrating the addition of Y-shaped adapters toDNA fragments and generation of subsets reducing the complexity of thesequences. In

FIG. 1A and FIG. 1B), Y-shaped adapters having non-complementarysequences on one end and appropriate overhangs for ligation on the otherend are ligated to DNA fragments. In FIG. 1C, a primer is annealed tothe denatured fragment-plus-adapter construct for use in single primerextension, PCR or other DNA polymerase reaction. The 5′-end of theprimer consists of a sequence complementary to the adapter region (b)and, at the 3′-end, the primer has one or more nucleotides (N) whichmust properly anneal to the fragment sequence in order for priming tooccur. Therefore, only a subset of fragment sequences that arecomplementary to the nucleotide(s) N of the primer will be replicated. Acapture moiety, in this case biotin, can be present to allow isolationof reaction products. In FIG. 1D, extension of the primer by DNApolymerase generates a product, the 3′-end of which is complementary tothe adapter region (a). Therefore, this DNA product can itself bereplicated by use of a primer complementary to the sequence (a) in aprimer extension, PCR, or other DNA polymerase reaction. Because of theY-shaped adapters, the products of such replication reactions will be innon-overlapping subsets defined by the nucleotide(s) N of the primer.The presence of a restriction endonuclease recognition site in theadapter, in this case GATC (FIG. 1D), allows for the release of any DNAproduct following capture by the moiety as shown in FIG. 1E.

FIG. 2A shows cDNA Sau3A1 fragments with Y shaped adapters andamplification with different primer pairs. FIG. 2B shows the results ofamplification with three different primer pairs.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides for the screening of complex DNA preparations,including complex DNA comprised of genomic segments or cDNAs, and theisolation of genes without requiring prior knowledge of theirbiochemical function or map position. Methods of the invention divideDNA into subsets and then manipulate the subsets using a mismatch repairsystem and capture techniques to obtain specific DNA sequences,including genomic subsets of long genomic DNA generated by selectiveamplification of sequences exhibiting low polymorphism.

As used herein, “polymorphism” refers to genetic sequence variationbetween different individuals of a species. A “homoduplex” isdouble-stranded DNA where both strands derive from the same genome orpools of genome samples, and a “heteroduplex” is double-stranded DNAwhere each strand originated from different genomes or different poolsof genomes. By “perfectly matched” is meant double-stranded DNA whereeach base residue is correctly paired with a base on the oppositestrand, i.e., A to T and C to G. By “mismatched” is meantdouble-stranded DNA where at least one base residue on either strand iseither not paired with any residue, or paired with an incorrect base,i.e., A not paired with T, C not paired with G.

In a typical practice of a method of the invention, at least one DNAsample is methylated, usually at the GATC sites with bacterial DAMmethylase, and the sample is then cut with an enzyme that makesinfrequent cuts such as Pvul (Nelson, S. F., et al., cited above). Anytype of DNA sample may be subjected to methods of the invention,including genomic DNA, genomic fragments, cDNA, cDNA fragments, andmixtures of any of these. It is an advantage of the invention that itcan be used to identify identical-by-descent sequences of lowpolymorphism in complex human or other genomic DNA samples. It can alsobe used to identify sequences of high polymorphism.

Adapters are then ligated to the fragments to obtainfragment-plus-adapter constructs. Linear or Y-shaped adapters may beemployed. Y-shaped adapters are used in many preferred embodiments, but,in some cases, where Y-shaped adapters are illustrated, the methods canalso be adapted to conventional linear adapters. Y-shaped adapters havebeen described (see Prashar, Y., and Weissman, S., Proc. Natl. Acad.Sci. USA, 1996, 93:659-663). A Y-shaped adapter typically has anoverhang on its 3′-end for ligation, and on the 5′-end, a stretch ofnoncomplementary sequence on the opposite strands, giving rise to itsY-shape (see FIG. 1A and B). It is an advantage of the invention that,in preferred embodiments, the Y-shaped adapters allow for the synthesisof non-overlapping subsets of DNA. In typical embodiments, if theinvention is carried out with conventional, linear primers, then thePCR-generated subsets will be partially overlapping, that is, some DNAsequences will be represented in more than one subset.

The fragment-plus adapter constructs are subjected to a PCR or to asingle primer extension reaction in the presence of a primercomplementary to at least a portion of the adapter at the 3′-end of thefragment-plus-adapter constructs and extending across the adapterligation, and having at least one nucleotide overlap into the DNAfragment sequence. As used herein, a “polymerase chain reaction”includes conventional PCR, as well as modifications employing betaine,proof-editing polymerases, DMSO, and the like, and combinations thereofLikewise, “rolling circle amplification” includes variants described byHingorani and O'Donnell, cited above, and specifically encompassesmodifications using a reconstituted bacterial polymerase III systemincluding holoenzyme, helicase, clamp proteins, and clamp loadingproteins (Bloom, L. B., et al., J. Biol. Chem., 1997, 272:27919-27930).

In some cases, a tag at the 5′-end, and a restriction endonucleaserecognition site at least about 6 nucleotides from the tag, are presentto allow capture of a DNA product and subsequent release by cutting withthe restriction endonuclease. In some embodiments, the annealed primerextends across the adapter ligation site one nucleotide into the DNAfragment sequence; in others, they extend two; and in others, more thantwo. The number of nucleotides, and the identity of the nucleotides thatthe primer extends across the adapter ligation site, determines themembers of the subset to be amplified. The tag in many embodiments isbiotin, illustrated in FIG. 1C.

In an alternative method for generating subsets, DNA samples are cutwith a restriction endonuclease, such as Bsl1, that generates variableoverhangs. That is, some of the bases in the recognition site can be ofany-two or more of the four possible bases G, A, T, or C. Adaptershaving overhangs complementary for this restriction endonucleaserecognition site are ligated onto the fragments. Adapter overhangshaving a unique sequence for the variable sites will only ligate to asubset of fragments that are complementary at those positions.Therefore, a subset of fragments will be replicated by a primercomplementary to the adapter. By employing Y-shaped adapters, thesubsets will be non-overlapping. Another advantage of this method isthat it is a simple process to ligate adapters of one sequence at oneend of the fragment and adapters of a second sequence at the other endof the fragment. If the adapters differ from each other in the primerannealing sequence of their non-complementary (Y-shaped) regions, thenit is possible to amplify only one strand of the duplex adapter-fragmentcomplex with the appropriate primer set in a PCR or other DNA polymerasereaction.

In a typical practice of a method of the invention, a subset offragments are generated from one sample of a DNA or a mixture of DNAs,and these are methylated. The same subset is obtained from a secondsample of DNA or mixture of DNAs, and these are not methylated. Mixing,denaturing and reannealing the methylated and unmethylated samplestogether generates hemimethylated heterohybrids, and, where a largenumber of DNA samples have been pooled together, most of the reannealedduplex DNA will be heterohybrids. The reannealing thus primarily resultsin perfectly matched heterohybrids or mismatched heterohybrids,depending upon the degree of polymorphism of the samples. In some cases,the mismatched heterohybrids are then selected by binding of MutS to themismatch or subjected to MutSLH, which nicks any that contain themismatched base pairs expected for regions of high polymorphism (seeU.S. Pat. No. 5,556,750 to Modrich, et al., Cheung, V. G., et al.,Nature Genetics, 1998, 18:225-230, and the references cited therein).

In the case where samples are treated with MutSLH, the nick that isgenerated in mismatched DNA is utilized to identify, isolate, amplify,or clone these fragments using a variety of methods that take advantageof the nick. In one case, a capture agent such as a biotin-taggednucleotide is added onto the nick by terminal transferase or some otherDNA polymerase and the nicked fragment is thereby isolated.Alternatively, the nicked strand can be removed by treatment with anexonuclease according to a published method (Nelson, S. F., et al.,cited above). The surviving strand is then selected by DNA amplificationor other methods. In another use of MutSLH nicked DNA, the 3′-OH of thenick serves as a primer for a DNA polymerase. Extension of the 3′-OHrequires that the DNA polymerase utilize a duplex DNA template by a nicktranslation or strand displacement reaction. The newly synthesized DNAcan be detected by the incorporation of a radioactively or fluorescentlylabelled nucleotide, or captured by the incorporation of a nucleotideappropriately tagged with a capture agent such as biotin. Also,extension of the nick where the Y-shaped adapter-fragment constructs ofthis invention are employed results in a DNA product which can bespecifically replicated with unique primer sets in a PCR reaction orwith a unique “splint oligonucleotide” in a rolling circleamplification. Referred to above, RCA is an isothermal amplificationreaction in which a DNA polymerase extends a primer on a circulartemplate (see Kornberg and Baker and other references cited above). Theproduct consists of tandemly linked copies of the complementary sequenceof the template.

In the case of RCA, the “splint oligonucleotide” is frequently asingle-stranded sequence complementary to the ends of the DNA thatresults from extension of the nick such that denaturation of the DNA andannealing of the splint to the extended strand circularizes it. If theDNA is circularized such that its two ends are brought together at anick, then the ends can be ligated together by DNA ligase forming acovalently closed circle. This DNA can then be amplified in an RCA.Another aspect of this invention is that DNA polymerase III holoenzymederived from E. coli or other bacteria, including gram-positive andgram-negative bacteria, or related DNA polymerases from eukaryotes thathave clamp (PCNA) and clamp loader (RFC) components (Kornberg and Baker,cited above) can be employed as the DNA polymerase in RCA. Use of DNApol III is advantageous in many embodiments because pol III has agreater rate and processivity than other DNA polymerases and providessuperior yield and ability to replicate long templates and templateshaving obstructions to DNA replication such as high GC content, orunfavorable secondary structure or sequence context. The E. coli dnaBand dnaC proteins or other helicases and the single-stranded DNA bindingprotein (SSB) can also be used to facilitate the reaction (Kornberg andBaker, cited above).

In another use of the nick generated in mismatched DNA by MutSLH, themismatched DNA is discarded and the perfectly matched DNA can thereby beselectively amplified. For example, PCR primers, or a splintoligonucleotide in the case of RCA, can be used to amplify those DNAsnot nicked by MutSLH whereas nicked DNA cannot provide an intact DNAtemplate.

The methods employed in this invention depend on the isolation ofheterohybrid DNA in which the two strands are derived from two differentDNA samples. This can be accomplished by published methods (Nelson, etal., cited above). Improved procedures that do not require methylationof fragment DNA are included in this invention. Sequences in theadapters are designed to allow selective cutting of homohybrid orheterohybrid DNA with restriction endonucleases. In some methods, theadapters contain two adjacent restriction enzyme recognition sites withspecific methylation patterns such that heterohybrid and homohybrid DNAscan be distinguished by the ability of the methyl groups to blockcutting by the restriction endonuclease. In other methods, partialrestriction endonuclease recognition sequences are present in which theadapter contains mismatched bases. In this case heterohybrid andhomohybrid DNAs can be distinguished by the elimination of themismatches which allows restriction endonuclease to cut these sites.

EXAMPLES Example 1. Procedure for creating cDNA or Genomic DNA Subsets

This example illustrates the use of PCR to amplify a subset of cDNA Themethod can be used also for total genomic DNA or other mixtures of DNAs.The Y shaped adapters are designed two create “butterfly” ends on theconstruct (see FIG. 2). The Y type adaptors enable only one strand butnot the other strand of the fragment to be amplified. Also, both strandscan be amplified separately. This is useful when the fragment contains amismatched base pair and it is desirable to amplify the strandsseparately. The Y shaped adapters also enable the amplified duplex to besequenced. The PCR primers are designed so that their 5′ end iscomplementary to adapter sequence, but 1-3 nucleotides at their 3′ end(designated by “N” in FIG. 2) must base pair with the target DNA insert.The target DNA sequences that will be amplified are determined by theidentity of the 3′ terminal nucleotides of the PCR primers. Therefore,only a subset of sequences will be amplified and the complexity of thesample will be reduced.

In this example, cDNA was cut with a 4 nucleotide-recognizingrestriction enzyme, Sau3AI. The restriction enzyme was inactivated afterdigestion was completed by treating it at 65C for 30 minutes. Thedigested DNA was then purified by phenol chloroform extraction. Y shapedadaptors were formed by annealing as follows: 1.3 nmol XS1, 1.3 nmolXS2, 5 mM Tris-HCI pH7.5 and 100 mM NaCI in 100 microliters volume at94C×10 min, cool down to 37C×2 hrs, then 32C×2 hrs, 30C×2 Hrs, 28×2 Hrs,25×2 Hrs and on ice. The fragments were then ligated to Y shapedadaptors as follows: cut cDNA 0.1 micrograms, adaptor pair 0.2micrograms /13 pmol, ligase 8u and 1X ligase buffer in 5 microliters at16C overnight. A subset of the sequences was then amplified with a pairof PCR primers (see primer sequences below) in the following mixture: 2microliters of 200-fold diluted ligated product from above, 2ul of 2micromolar each primer, 0.75 units AmpliTag Gold DNA Polymerase (PerkinElmer), 2 mM each dNTP and 1X DNA polymerase buffer supplied by themanufacturer. The PCR was done in a Perkin Elmer Cetus Gene Amp PCRSystem 9600 with the program:

95C, 4min

five cycles

94C, 30 sec

55C, 30 sec

72C, 30 sec

25 cycles

94C, 30 sec

65C, 30 sec

72C, 30 sec

72C, 5 min.

As indicated in FIG. 2, three different primer sets were used. Wellnumber 1 contained a primer sequence which has C and G as the two 3′terminal nucleotides (designated PS1CG), and PS2AG which has A and Gasthe terminal nucleotides. Wells 2 and 3 of FIG. 2 contained primer setswith different terminal nucleotides as indicated. As expected, the threePCR reactions produced different band patterns determined by whichprimer set was used.

Adaptor for Sau3AI cutting

XS1 (22 nt )

CGTCCGGCGCCAGCGACGGTCAG—SEQ ID No: 1

XS2 (29 nt)

GATCCTGACCGTCCATCTCTGTCGCAGCG—SEQ ID No: 2

PCR Primers (corresponding to above Sau3AI adaptors):

Set1:

PS1CG (28 nt)

CGTCCGGCGCAGCGACGGTCAGGATCCG—SEQ ID No: 3

PS2AG (31 nt)

CGCTGCGACAGAGATGGACGGTCAGGATCAG—SEQ ID No: 5

Set2:

PS1CGT (29 nt)

CGTCCGGCGCAGCGACGGTCAGGATCCGT—SEQ ID No: 5

PS2AGG0 (32 nt)

CGCTGCGACAGAGATGGACGGTCAGGATCAGG—SEQ ID No: 6

Set3:

PS1CGT (29 nt)

CGTCCGGCGCAGCGACGGTCAGGATCCGT—SEQ ID No: 7

PS2TCG (32 nt)

CGCTGCGACAGAGATGGACGGTCAGGATCTCG—SEQ ID No: 8

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. For example, there are numerous variations of steps in theoverall procedures, and for preparing the probes. Variations in primershaving larger overlap with DNA fragments and various amplificationtechniques, for example, have already been mentioned. Followingselective isolation of duplex DNA, it could be transcribed with T7 orother appropriate RNA polymerase, and the RNA used as a direct probe, orreconverted into double-stranded DNA in some embodiments. It isintended, however, that all such obvious modifications and variations beincluded within the scope of the present invention, which is defined bythe following claims. The claims are intended to cover the claimedcomponents and steps in any sequence which is effective to meet theobjectives there intended, unless the context specifically indicates thecontrary.

The papers, books and patents cited herein are expressly incorporated intheir entireties by reference.

8 1 22 DNA Artificial Sequence Description of Artificial Sequence DNAadaptor for cutting by Sau3A1 restriction endonuclease. 1 cgtccggcgcagcgacggtc ag 22 2 29 DNA Artificial Sequence Description of ArtificialSequence DNA adaptor for cutting by Sau3A1 restriction endonuclease. 2gatcctgacc gtccatctct gtcgcagcg 29 3 28 DNA Artificial SequenceDescription of Artificial Sequence PCR primer corresponding DNA adaptorshown in SEQ ID NO1. 3 cgtccggcgc agcgacggtc aggatccg 28 4 31 DNAArtificial Sequence Description of Artificial Sequence PCR primercorresponding to DNA adaptor shown in SEQ ID NO2. 4 cgctgcgacagagatggacg gtcaggatca g 31 5 29 DNA Artificial Sequence Description ofArtificial Sequence PCR primer corresponding to DNA adaptor shown in SEQID NO1. 5 cgtccggcgc agcgacggtc aggatccgt 29 6 32 DNA ArtificialSequence Description of Artificial Sequence PCR primer corresponding toDNA adaptor shown in SEQ ID NO2. 6 cgctgcgaca gagatggacg gtcaggatca gg32 7 29 DNA Artificial Sequence Description of Artificial Sequence PCRprimer corresponding to DNA adaptor shown in SEQ ID NO1. 7 cgtccggcgcagcgacggtc aggatccgt 29 8 32 DNA Artificial Sequence Description ofArtificial Sequence PCR primer corresponding to DNA adaptor shown in SEQID NO2. 8 cgctgcgaca gagatggacg gtcaggatct cg 32

What is claimed is:
 1. A method for isolating DNA subsets from a firstsample and a second sample for use in genomic mismatch analysis,comprising: (a) subjecting first portions of said first and secondsamples separately to a first oligonucleotide consisting of at least twocopies of any dinucleotide, trinucleotide, or tetranucleotide repeatattached to a support to obtain first and second sample fractions thatbind to the first oligonucleotide; (b) subjecting second portions ofsaid first and second samples separately to a second oligonucleotideconsisting of at least two adjacent copies of any dinucleotide,trinucleotide or tetranucleotide repeat different than that employed instep (a) attached to a support to obtain first and second samplefractions that bind to the second oligonucleotide; (c) mixing the firstsample fraction obtained in step (a) with the second sample fractionobtained in step (b), and mixing the second sample fraction obtained instep (a) with the first sample fraction obtained in step (b); (d)denaturing and reannealing each mixture to form heterohybrids, whereinthe heterohybrids comprise the DNA subsets to be used in genomicmismatch analysis.
 2. The method according to claim 1 further comprisingthe step of subjecting the DNA subsets to genomic mismatch analysis. 3.A method according to claim 2 wherein the first oligonucleotide consistsof at least two CA dinucleotide repeats, and the second oligonucleotideconsists of at least two GT dinucleotide repeats.
 4. A method accordingto claim 1 comprising the further step of subjecting the heterohybridsto allele frequency differences analysis.
 5. A method according to anyof claims 2, 3, or 4 comprising the further step of capturing mismatchedDNA subsets by incubating the subsets with biotinylated MutS attached toa support bearing avidin or strepavidin.
 6. A method according to any ofclaims 2, 3, or 4 wherein the first and second samples comprise genomicDNA, genomic DNA fragments, or mixtures thereof.
 7. A method accordingto claim 6 wherein the DNA subsets are used to identify genomic IBDsequences.
 8. A method according to any of claims 2, 3, or 4 wherein thefirst and second samples comprise cDNA.
 9. A method according to claim 8wherein the samples are analyzed for differences by separation of therecovered cDNA fragments using techniques selected from the groupconsisting of sequencing gels, capillaries, mass spectroscopy, labellingwith radioisotopes, labelling with antigenic epitopes, labelling withfluorescent dyes, mixing and hybridizing to arrays of genomic DNA clonesand combinations of any of these procedures.
 10. A method according toclaim 8 wherein the samples are analyzed for differences by separationof the recovered cDNA fragments using techniques selected from the groupconsisting of sequencing gels, capillaries, mass spectroscopy, andcombinations of any of these separation techniques.
 11. A methodaccording to claim 8 wherein the recovered cDNA fragments are labelledwith radioisotopes, antigenic epitopes or fluorescent dyes.
 12. A methodaccording to claim 8 wherein the cDNA samples are used to detecthomozygosity for mutations in affected subjects, and heterozygosity incarriers.
 13. A method according to claim 12 wherein cDNA samples areprepared from homozygous-affected subjects in a family and from one orboth parents.
 14. A method according to claim 8 wherein the cDNA samplesare used to detect new mutations in a subject with de novo dominantmutation, where the parents of the subject do not show the disorder, byanalysis of cDNA displays prepared from the subject cDNA, from eachsubject parent's cDNA, from heterohybrids between subject and parentcDNA, and from heterohybrids prepared between the cDNA of the twoparents.
 15. A method according to claim 8 wherein the cDNA samples areused to detect mutations in cDNA in cancers that are not present in cDNAof normal tissue from the same subject.
 16. A method according to claim8 which analyzes differences in the relative amounts of heterozygosityin cDNA fragments from control subjects, and from subjects with agenetic disorder of unknown pattern of inheritance or a polygenicdisorder.
 17. A method according to claim 14 wherein mRNA from a groupof control subjects and from a group of subjects with the disorder areprepared, cDNA generated from both RNA groups are prepared, and cDNAdisplays are analyzed with subject pools, control pools, and pools fromheterohybrids between subjects and controls.